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Method for purifying albumin

a technology of purification method and albumin, which is applied in the field of purification method, can solve the problems of time-consuming and/or expensive, and the purification process is required

Active Publication Date: 2012-06-14
ALBUMEDIX LIMITED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0008](iii) eluting the albumin, a variant or fragment thereof, a fusion protein comprising albumin, a variant or fragment thereof, or a conjugate comprising albumin, a variant or fragment thereof from the matrix to provide a purified albumin, a variant or fragment thereof, a fusion protein comprising albumin, a variant or fragment thereof, or a conjugate comprising albumin, a variant or fragment thereof.The inventors have identified that well known separation steps, previously used in purification of albumins, such as cation exchange and dye binding processes; can be replaced by a single affinity process which increases clearance of undesired proteins relative to such steps and / or increases the yield.

Problems solved by technology

A problem with current albumin products is the purification process required.
High purity can be achieved but this requires multiple chromatographic purification steps which can be time consuming and / or expensive.

Method used

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  • Method for purifying albumin
  • Method for purifying albumin
  • Method for purifying albumin

Examples

Experimental program
Comparison scheme
Effect test

example 1

Comparison of rHSA 2 Step AlbuPure™ Purification and 3 Step Conventional Purification

[0069]The conventional 3 step purification of cation ion exchange, anion exchange and dye affinity chromatography was performed as described in WO 2000 / 044772.

[0070]The 2 step AlbuPure™ purification was performed by conditioned the rHSA fermentation supernatant for chromatography using acetic acid and water to achieve a pH of approximately 5.3 and a conductivity of approximately 3 mS·cm−1. A column packed with AlbuPure™ matrix was equilibrated with 50 mM sodium acetate pH 5.3. A volume of conditioned sample equivalent to 20 mg rHSA·mL−1 matrix was loaded. The matrix was washed sequentially with 50 mM sodium acetate pH 5.3, 50 mM sodium phosphate pH 6.0, 50 mM sodium phosphate pH 7.0 and 50 mM ammonium acetate pH 8.0 before being eluted with 50 mM ammonium acetate, 10 mM sodium octanoate pH 7.0 and finally regenerated with 0.5M sodium hydroxide. The AlbuPure™ eluate was conditioned for anion ion exch...

example 2

rHSA Fusions

[0072]Yeast derived culture supernatant containing c-terminal rHSA fusions of prosaptide, T20 (PCT / 1B03 / 00434) and IL1RA were conditioned for chromatography using acetic acid and water to achieve a pH of approximately 5.3 and a conductivity of between 2.6 and 3.3 mS·cm−1. A 1.6 cm×11.0 cm (22.1 mL) column packed with AlbuPure™ matrix was equilibrated with 50 mM sodium acetate pH 5.3. A volume of conditioned sample equivalent to 20 mg fusion protein·mL−1 matrix was loaded. The matrix was washed sequentially with 50 mM sodium acetate pH 5.3, 50 mM sodium phosphate pH 6.0, 50 mM sodium phosphate pH 7.0 and 50 mM ammonium acetate pH 8.0 before being eluted with 50 mM ammonium acetate, 10 mM sodium octanoate pH 7.0 and finally regenerated with 0.5M sodium hydroxide. SDS_PAGE of Prosaptide albumin fusion purification is shown in FIG. 1. SDS-PAGE of T20 albumin fusion purification is shown in FIG. 2. SDS-PAGE of IL1RA Albumin fusion purification is shown in FIG. 3.

example 3

Anti FITC (scFv(vHvL)-rHSA-FLAG) Antibody

[0073]Yeast derived culture supernatant containing the anti FITC (scFv(vHvL)-rHSA-FLAG) (disclosed in EP application No 09 159 642) antibody fusion (N-terminal fusion) was conditioned for chromatography using water to achieve a pH of 6.2 and a conductivity of 7.9 mS·cm−1. A 4.4 cm×11.0 cm (167.3 mL) column packed with AlbuPure™ matrix was equilibrated with 50 mM sodium phosphate pH 6.0. A volume of conditioned sample equivalent to 9.5 mg protein·mL−1 matrix was loaded. The matrix was washed sequentially with 50 mM sodium phosphate pH 6.0, 50 mM sodium phosphate pH 7.0 and 50 mM ammonium acetate pH 8.0. Bound protein was eluted first with 50 mM ammonium acetate, 10 mM sodium octanoate pH 7.0 and then with 50 mM ammonium acetate, 30 mM sodium octanoate, 200 mM sodium chloride pH7.0. The matrix was regenerated with 0.5M sodium hydroxide. A total of 84% of the anti FITC (scFv(vHvL)-rHSA-FLAG) was recovered in the 2 eluates combined, as estimated ...

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Abstract

An improved method for purifying albumin, a variant or fragment thereof, a fusion protein comprising albumin, a variant or fragment thereof, or a conjugate comprising albumin, a variant or fragment thereof is disclosed.

Description

REFERENCE TO SEQUENCE LISTING[0001]This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.FIELD OF THE INVENTION[0002]The present invention relates to a method for purifying albumin, a variant or fragment thereof, a fusion protein comprising albumin, a variant or fragment thereof, or a conjugate comprising albumin, a variant or fragment thereof. The albumin may be a serum albumin, such as human serum albumin, obtained from an animal or from a microorganism such as a yeast.BACKGROUND OF THE INVENTION[0003]Albumin is used to treat patients with severe burns, shock or blood loss. It is also used to supplement media used for growing higher eukaryotic cells and as an excipient for pharmacologically active compounds, many of which need to be stabilised. Albumin fusion proteins are a fusion of a protein to albumin, or to a variant or fragment thereof, and increases the half life of the protein, for example incr...

Claims

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Application Information

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IPC IPC(8): C07K1/22
CPCC07K14/765C07K16/00C07K16/18C07K16/44C07K2319/43C07K2317/569C07K2317/622C07K2319/20C07K2319/31C07K2317/41
Inventor BLACKWELL, LEE EDWARDCAMERON, JASONMORTON, PHILLIP HARVEY
Owner ALBUMEDIX LIMITED
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